The human T-cell leukemia virus type 1 oncoprotein Tax is a phosphoprotein with a predominately nuclear subcellular localization that accomplishes multiple functions via protein-protein interactions. It has been proposed that regulation of this protein's pleiotropic functions may be accomplished through phosphorylation of specific amino acid residues. We have conducted a phosphoryl mapping of mammalian-expressed Tax protein using a combination of affinity purification, liquid chromatography tandem mass spectrometry, and site-directed substitution mutational analysis. We achieved physical coverage of 77% of the Tax sequence and identified four novel sites of phosphorylation at Thr-48, Thr-184, Thr-215, and Ser-336. Previously identified potential serine phosphorylation sites at Ser-10, Ser-77, and Ser-274 could not be confirmed by mass spectrometry. The functional significance of these novel phosphorylation events was evaluated by mutational analysis and subsequent evaluation for activity via both CREB and NF-kappaB-responsive promoters. Our results demonstrate that phosphorylation at Thr-215 is associated with loss of both Tax functions, phosphorylation at Thr-48 was specifically deficient for activation via NF-kappaB, and phosphorylation at Thr-184 and Ser-336 had no effect on these Tax functions. Semiquantitation of phosphopeptides revealed that the majority of Tax was phosphorylated at Thr-48, Thr-184, Thr-215, and Ser-336, whereas only a minor population of Tax was phosphorylated at either Ser-300 or Ser-301. These results suggest that both positive and negative phosphorylation signals result in the maintenance of a subfraction of Tax as "active" protein.